British Journal of Pharmacology (2010), 160, 523529 2010 The Authors Journal compilation 2010 The British Pharmacological

al Society All rights reserved 0007-1188/10 www.brjpharmacol.org

THEMED ISSUE: CANNABINOIDS REVIEW Phytocannabinoids beyond the Cannabis plant do they exist?
bph_745 523..529

Jrg Gertsch1, Roger G Pertwee2 and Vincenzo Di Marzo3

Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland, 2School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, UK, and 3Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, NA, Italy It is intriguing that during human cultural evolution man has detected plant natural products that appear to target key protein receptors of important physiological systems rather selectively. Plants containing such secondary metabolites usually belong to unique chemotaxa, induce potent pharmacological effects and have typically been used for recreational and medicinal purposes or as poisons. Cannabis sativa L. has a long history as a medicinal plant and was fundamental in the discovery of the endocannabinoid system. The major psychoactive Cannabis constituent D9-tetrahydrocannabinol (D9-THC) potently activates the G-protein-coupled cannabinoid receptor CB1 and also modulates the cannabinoid receptor CB2. In the last few years, several other non-cannabinoid plant constituents have been reported to bind to and functionally interact with CB receptors. Moreover, certain plant natural products, from both Cannabis and other plants, also target other proteins of the endocannabinoid system, such as hydrolytic enzymes that control endocannabinoid levels. In this commentary we summarize and critically discuss recent ndings.
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British Journal of Pharmacology (2010) 160, 523529; doi:10.1111/j.1476-5381.2010.00745.x

This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x
Keywords: phytocannabinoid; cannabinoid; plant natural products; Cannabis; endocannabinoid system Abbreviations:

CB1, type-1 cannabinoid receptor; CB2, type-2 cannabinoid receptor; CP55940, ()-cis-3-[2-hydroxy-4-(1,1dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; D9-THC, D9-tetrahydrocannabinol; DIM, 3,3diindolylmethane; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; FDA, US Food and Drug Administration; Gi/o, G-protein alpha subunit; GPR55, orphan receptor G-protein-coupled receptor 55; MAGL, monoacylglycerol lipase; NAEs, N-acylethanolamines; PPAR, peroxisome proliferator-activated protein; SR144528, (1S-endo)-5-(4-Chloro-3-methylphenyl)-1-((4-methylphenyl)methyl)-N-(1,3,3trimethylbicyclo(2.2.1)hept-2-yl)-1H-pyrazole-3-carboxamide; SR141716A, N-(piperidin-1-yl)-5-(4chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; Tmax, time to maximal concentration in plasma (pharmacokinetic parameter); TRPV1, transient receptor potential vanilloid-1 receptor; WIN55212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4benzoxazin-6-yl]-1-naphtaleneylmethanone

Today we perceive the endocannabinoid system (ECS) as a rather complex lipid signalling network in which different proteins play distinct roles in the control or in the modulation of numerous physiological and pathophysiological

Correspondence: Jrg Gertsch, Institute of Biochemistry and Molecular Medicine, Bhlstrasse 28, University of Bern, 3012 Bern, Switzerland. E-mail: gertsch@mci.unibe.ch The molecular receptor nomenclature used throughout this commentary conforms to the BJPs Guide to Receptors and Channels (Alexander et al., 2009). Received 12 January 2010; revised 18 February 2010; accepted 23 February 2010

processes (Pertwee, 2005; Di Marzo, 2008). The ECS comprises classical cannabinoid receptors (CB1 and CB2), potentially also the orphan receptor GPR55, and arachidonic acid-derived ligands, which, however, also promiscuously target other receptors like, e.g. TRPV1 and PPAR-gamma (OSullivan, 2007; De Petrocellis and Di Marzo, 2010; Ross, 2009; Pertwee, 2010). Importantly, the enzymes degrading the endocannabinoids anandamide and 2-arachidonoyl glycerol, namely fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), have been shown to be promising therapeutic targets (Di Marzo, 2008). Finally, there appears to be an anandamide cellular reuptake mechanism that can be blocked by specic

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inhibitors (Di Marzo, 2008). Both cannabinoid receptor agonists and antagonists have actual or potential therapeutic applications (Di Marzo, 2008; Oesch and Gertsch, 2009; Pertwee, 2009). Cannabinoids are dened as the terpenophenolic constituents of Cannabis sativa L and until recently, the phenylterpenoid D9-THC and some of its naturally occurring derivatives were the only plant natural products known to directly interact with cannabinoid receptors. However, in the last few years, several non-cannabinoid plant natural products have been reported to act as cannabinoid receptor ligands. This prompts us to dene phytocannabinoids as any plant-derived natural product capable of either directly interacting with cannabinoid receptors or sharing chemical similarity with cannabinoids or both. Direct cannabinoid receptor ligands are compounds that show high binding afnities (in the lower nM range) for cannabinoid receptors and exert discrete functional effects (i.e. agonism, neutral antagonism or inverse agonism). By contrast, indirect ligands target either key proteins within the ECS that regulate tissue levels of endocannabinoids or allosteric sites on the CB1 receptor. Certain plant natural products, including some cannabinoids, possess at least some of these properties. Given the often high variability of molecular pharmacological data obtained in different laboratories and the distinct degrees of scrutiny of the experimental setup, molecular pharmacological data on natural products should always be interpreted with care (Gertsch, 2009). For example, the availability of CB receptor KO mice provides a powerful means of investigating the actual cannabimimetic nature of a particular compound in vivo. This commentary focuses on natural products from medicinal and dietary plants which have been reported to interact with the ECS.

2009). Different Echinacea N-isobutylamides are orally bioavailable resulting in nM plasma levels in humans (Woelkart et al., 2008). The polyacetylenic polyyne falcarinol, which is found in different plants of the Apiaceae family (e.g. in carrots) shows signicant binding interactions with both cannabinoid receptors, but appears to selectively undergo an alkylation reaction with the CB1 receptor (Ki value <1 mM), leading to relatively potent inverse agonistic and proinammatory effects in human skin (Leonti et al., 2010). Finally, it has been proposed that certain dietary fatty acids, which can also be found in plants, can modulate the ECS by inuencing the availability of phospholipid biosynthetic precursors of endocannabinoids (Banni and Di Marzo, 2009).

Terpenes
The bicyclic sesquiterpene, b-caryophyllene (trans-isomer) (Table 2), which is a plant volatile very frequently found in plants, has been shown to selectively target the CB2 receptor at nM concentrations (Ki = 155 nM) and to act as a full agonist (Gertsch et al., 2008). Remarkably, b-caryophyllene is also a major compound in Cannabis sativa L. essential oil. Thus, Cannabis produces two entirely different chemical scaffolds able to differentially target CB receptors. While studies on the pharmacokinetics of b-caryophyllene are still ongoing, it is already clear that this cyclobutane-ring containing terpene is readily bioavailable, and, unlike many polyphenolic natural products, is not metabolized immediately but shows a Tmax >1 h after one single oral administration (J.G., unpublished data). Orally administed b-caryophyllene (<5 mgkg-1) produces strong anti-inammatory and analgesic effects in wildtype mice but not in CB2 receptor knockout mice, which is a clear indication that it may be a functional CB2 ligand. Ongoing studies show that b-caryophyllene is effective at reducing neuropathic pain in a CB2 receptor-dependent manner (Zimmer et al., 2009). Therefore, the FDA approved food additive b-caryophyllene has the potential to become an attractive candidate for clinical trials targeting the CB2 receptor (Gertsch, 2008). Interestingly, the diterpene salvinorin A from Salvia divinorum Epling & Jativa-M (Table 1) has been reported to be a selective high-afnity kappa-opioid receptor (KOP) agonist, but recent data also suggest that it may interact with a putative CB receptor/KOP heterodimer which may be formed during inammatory conditions (Fichna et al., 2009). To date, binding experiments have shown that salvinorin A has very low afnity for homomeric cannabinoid receptors and does not inhibit endocannabinoid degradation (Capasso et al., 2008). Consequently, further research is needed to establish whether salvinorin A interacts with a putative cannabinoid/KOP heterodimeric receptor or whether the cannabimimetic effects reported are indirectly mediated via KOP. More recently, two naturally occurring quinonoid triterpenoids, pristimerin (Table 1) and euphol, were found to inhibit MAGL with high potency (IC50 = 93 nM and 315 nM respectively) through a reversible mechanism (King et al., 2009). As this class of triterpenes is relatively frequent in nature, it may not be unusual to nd indirect rather than direct agonists of cannabinoid receptors among plant secondary metabolites. Several distinct triterpenes are known to modulate immune

Fatty acid derivatives

Despite the fact that N-acylethanolamines (NAEs) (Table 1) from plants do not interact with CB receptors (plants do not generally produce arachidonic acid, which is the acyl scaffold favoured for CB interaction) they have been shown to inhibit FAAH, thus leading to an increase in endocannabinoid tone. N-linoleoylethanolamide and N-oleoylethanolamide, which are found not only in chocolate (Theobroma cacao L.) but also other plants (Di Marzo et al., 1998), and the widespread NAE palmitoylethanolamide, inhibit anandamide breakdown (Maurelli et al., 1995; Di Tomaso et al., 1996). Certain N-alkylamides (alkamides) from Echinacea spp. (Table 2) have been shown to interact functionally with the human CB2 receptor with low nM to mM Ki values (Gertsch et al., 2006). These N-isobutylamides selectively act at the CB2 receptor over the CB1 receptor, leading to an increase in intracellular calcium which could be blocked by the selective CB2 receptor inverse agonist SR144528, but they do not modulate the Gai signalling pathway. Intriguingly, CB2 receptor-binding N-alkylamides show similar anti-inammatory effects as anandamide (e.g. inhibition of TNF-a) at low nM concentrations (Raduner et al., 2006). Certain Echinacea N-alkylamides inhibit anandamide reuptake in vitro (Chicca et al., 2009). Like anandamide, N-alkylamides also target PPAR-gamma (Spelman et al.,
British Journal of Pharmacology (2010) 160 523529

Table 1
Origin CB receptor afnity Function In vivo efcacy Other targets (ECS) GPR55 References

Plant natural products that have been suggested to exert cannabimimetic effects but do not interact directly with cannabinoid (CB) receptors

Structure

Name

N-acylethanolamines

Widespread in plants

No afnity

OH N H R = linoleoyl; oleoyl; palmitoyl

FAAH inhibitors Indirect cannabimimetics

Validated in CB1 and CB2 KO mice

Maurelli et al., 1995; Di Tomaso et al., 1996; Di Marzo, 2008 Capasso et al., 2008; Fichna et al., 2009;

Salvinorin A

Salvia divinorum Epling & Jativa-M

Insignicant afnity to CB receptors

No data

KOP agonist

Indirect cannabimimetic effects at CB1 (mechanism unknown)

CO2 CH3

Pristimerin

Relatively widespread in the Celastraceae

No data

Potent reversible MAGL inhibitor (IC50 value <100 nM)

No data

No data

King et al., 2009

HO

OH

Kaempferol

Widespread in plants

No afnity

No data

No data

Thors et al., 2007; 2008

HO

FAAH inhibitor (IC50 value <1 mM)

OH

OH

OH

Trans-resveratrol

Insignicant afnity

Insignicant effects

No data

No data

Prather et al., 2009

HO

Relatively widespread in plants (e.g. Vitis vinifera L.) Curcuma spp. Insignicant afnity

OH

H3CO

OCH 3

Curcumin

Insignicant effects

No data

No data

Prather et al., 2009

HO

OH

OH

Phytocannabinoids beyond the Cannabis plant J Gertsch et al

OH

Epigallocatechin3-O-gallate

Insignicant afnity

No data

No data

No data

Korte et al., 2010

HO

OH

Relatively widespread in plants (e.g. Camellia sinensis L.)

OH

OH

OH

OH

ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase.

525

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Table 2
Origin Cannabis sativa L. CB receptor afnity Function In vivo efcacy Other targets (ECS)

Plant natural products that have been shown to interact directly with cannabinoid (CB) receptors
References Mechoulam, 1986; Pertwee, 2006

Structure

Name

D9-THC Non-selective CB1 and CB2 afnity (Ki values <50 nM) (human) Parial agonist Gi/o Inhibition by SR141716 and SR144528 Validated in CB1 and CB2 KO mice GPR55 PPARs Different ion channels

OH

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Echinacea spp. No data Selective CB2 afnity (Ki value <100 nM) (human) Parial agonist [Ca2+] i Inhibition by SR144528 No data Parial agonist [Ca2+]i Inhibition by SR144528 Raduner et al., 2006; Chicca et al., 2009 Echinacea spp. Selective CB2 afnity (Ki value <100 nM) (human) Selective CB2 afnity (Ki value <200 nM) (human) Full agonist Gi/o [Ca2+]i Validated in CB2 KO mice Widespread in plants PPAR-g Inhibition of AEA transport Partial FAAH inhibtion PPAR-g Inhibition of AEA transport Parial FAAH inhibition No data Raduner et al., 2006; Chicca et al., 2009 Gertsch et al., 2008 Relatively widespread in Apiaceae (e.g. Daucus carota L.) Non-selective CB1 afnity (Ki value <1 mM) (human) Ruta graveolens L. CB1 receptor-selective inverse (covalent) agonist Inhibition of AEA/ WIN55212-2 No data No data No data Leonti et al., 2010 No data No data Rollinger et al., 2009 Selective CB2 afnity (Ki value <10 mM) (human) Relatively widespread in the Brassica genus Selective CB2 afnity (Ki value @1 mM) (human) Partial agonist at CB2 receptor No data No data Yin et al., 2009

N-alkylamide

N H

N-alkylamide

N H

Phytocannabinoids beyond the Cannabis plant J Gertsch et al

b-caryophyllene

HO

Falcarinol

Rutamarin

NH

N H

DIM 3,3-diindolylmethane metabolite from indole-3-carbinol

D9-THC is shown as the major phytocannabinoid from Cannabis sativa L. but there are several other structurally related cannabinoids that interact with CB receptors. D9-THC, D9-tetrahydrocannabinol; DIM, 3,3-diindolylmethane; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; PPAR, peroxisome proliferator-activated protein.

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527

functions through yet unknown mechanisms (Rios, 2010) and it will thus be interesting to see in a more systematic study whether other similar triterpenoids are also able to inhibit MAGL.

Polyphenols
The dietary polyphenols trans-resveratrol and curcumin (Table 1) were reported to bind selectively to the human CB1 cannabinoid receptor with low nM Ki values (5.9 nM and 45 nM respectively) and to exert potent pharmacological effects in mice similar to those induced by the CB1 receptor inverse agonist rimonabant (Seely et al., 2009). Intrigued by this unexpected nding, our research groups independently measured the binding afnities of these compounds for CB1 and CB2 receptors in our laboratories. In our experiments, trans-resveratrol and curcumin only displaced [3H]CP55 940 from cannabinoid receptors at high mM concentrations, suggesting that they lack signicant afnity for these receptors. Also polydatin, a glycosilated form of resveratrol, was inactive in these binding assays. Recently, the senior author of the original report retracted the paper (Prather et al., 2009). Hence, neither trans-resveratrol nor curcumin interact functionally with the CB1 receptor, despite the fact that these compounds appear to share the ability of the CB1 receptor inverse agonist, rimonabant, to induce weight loss in mice. More recently, catechin-derivatives were shown to bind to human cannabinoid receptors rather non-selectively at high mM concentrations (Korte et al., 2010). Among these, epigallocatechin 3-gallate and (-)-epigallocatechin (Table 1) were reported to bind to the CB1 receptor with Ki values of 33.6 and 35.7 mM respectively. However, these Ki values may not be correct. For the calculation of the Ki values the Cheng-Prussof equation (Ki = IC50/1+([S]/Kd) was not applied correctly. The EC50 values used to calculate the Ki values were approximations as neither compound produced more than 60% radioligand displacement even at the highest concentration used. Catechins are very widespread plant secondary metabolites which may provide nutritional health benets. The same group has recently reported similar CB1 and CB2 receptor Ki values for delphinidin and cyanidin, two hydrophilic anthocyanidins (Korte et al., 2009). In both reports, no functional data were shown. In our hands, avonoid-type compounds (catechins, anthocyanidins, avones) lead to negligible or very high Ki values, which likely reect a nonspecic molecular denaturation of the protein surface rather than a functional binding interaction. Similar potentially artefactual effects would most likely be observed with other GPCRs. Plant polyphenols, such as phenylpropanoids (e.g. epigallocatechin 3-O-gallate, curcumin, resveratrol) possess chemical scaffolds which at mM concentrations bind to protein targets in vitro with limited specicity. This is clearly reected by numerous reports on protein binding interactions that such compounds undergo in the mM range (Anand et al., 2008; Bisht et al., 2009). At the macroscopic level, polyphenols (i.e. tannins) have been used to tan leather by denaturing of proteins, and at the microscopic level mM concentrations of polyphenols interact with multiple protein binding sites (via their hydroxyl groups) non-specically and therefore such

compounds score as frequent hitters in vitro. The great majority of established cannabinoid receptor ligands are highly lipophilic, which reects the nature of the active site within cannabinoid receptors. Thus, hydrophilic polyphenols like catechins and anthocyanidins would clearly be atypical cannabinoid receptor ligands. More interesting are ndings that certain avonoids inhibit fatty acid amide hydrolase (FAAH), which is the enzyme responsible for the breakdown of the endogenous CB receptor ligand anandamide (Thors et al., 2007; 2008). Both the isoavonoid genistein and the avonoids kaempferol (Table 1), 7-hydroxyavone and 3,7-dihydroxyavone have been shown to concentration-dependently inhibit anandamide hydrolysis in rat brain homogenates, albeit at relatively high concentrations (IC50 values between 2 and 10 mM). Nevertheless, the authors of these studies showed a preliminary structure-activity relationship with 7-hydroxyavone being the most potent inhibitor (IC50 value <1 mM). An abundant literature is devoted to mechanisms underlying the biological activity of plant polyphenols (LandisPiwowar and Dou, 2008; Bisht et al., 2009). However, although most benecial and potentially therapeutic effects of trans-resveratrol, curcumin, catechins and kaempferol-type avonoids are typically detected in the low mM range in vitro, all such compounds show limited bioavailability and poor pharmacokinetics in vivo with reported plasma concentrations in the low nM range (DuPont et al., 2004; Garcea et al., 2004; Boocock et al., 2007).

Other plant natural products with binding afnity to the CB2 receptor
Other plant natural products have been shown to bind weakly to the CB2 receptor. These include the coumarin derivative rutamarin from the medicinal plant Ruta graveolens L. (Rollinger et al., 2009) and 3,3-diindolylmethane (DIM) (Table 2), which is an anticarcinogenic metabolite generated by ingestion of indole-3-carbinol. Indole-3-carbinol is commonly found in Brassica vegetables. DIM has been shown to be a weak CB2 receptor partial agonist (Yin et al., 2009).

Conclusions
There is no doubt that phytocannabinoids from Cannabis have greatly inuenced research on the ECS and without the milestone discovery that D9-THC is the main psychoactive principle (reviewed in Mechoulam, 1986) many of the subsequent discoveries in the eld of cannabinoid research would probably not have been made. Furthermore, with the development of therapeutic Cannabis extracts, as with Sativex, this plant is also likely to provide new pharmaceutical applications in the future. The question remains as to why Cannabis sativa L. appears to be the only plant that produces a metabolite (D9-THC acid) that readily leads to its decarboxylation product D9-THC, which is the most potent phytocannabinoid activator of the CB1 receptor. Interestingly enough, while nature may have been rather parsimonious in its provision of botanical secondary metabolites that activate the
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Phytocannabinoids beyond the Cannabis plant J Gertsch et al

CB1 receptor, there is an increasing number of plant-derived natural products reported to target the CB2 receptor to varying degrees. Flavonoids, which belong to natural polyphenols that readily interact with proteins, may target some of the proteins within the ECS, such as FAAH. However, no convincing evidence has been provided that polyphenols modulate cannabinoid receptors with signicant potency. The nding that certain triterpenes potently inhibit MAGL further adds to the repertoire of plant-produced indirect cannabinoid receptor agonists. Although higher plants do not contain endocannabinoids and lack the classical G-protein-coupled cannabinoid receptors, they do express enzyme isoforms that resemble some of the enzymes known to be important in the processing of endocannabinoids (Shrestha et al., 2006). Plants produce fatty acid amides, some of which are able to inhibit the degradation of anandamide but do not generally bind with signicant afnity to CB receptors (Gertsch et al., 2006; Di Marzo et al., 2007). At present, the only phytocannabinoid that has been discovered to also exist in plants other than Cannabis is b-caryophyllene, which is among the most abundant plant essential oil components. Although D9-THC is a partial agonist at both CB1 and CB2 receptors, it has signicant lower efcacy at the CB2 receptor. Another phytocannabinoid, D9-tetrahydrocannabivarin, has also recently been shown to be a CB2 receptor partial agonist, but is also a CB1 receptor antagonist (Bolognini et al., 2010). Therefore, b-caryophyllene, which is also one of the most abundant secondary metabolites in Cannabis essential oil, could be considered as a true CB2 receptor-selective Cannabis constituent. During mammalian evolution contacts with direct CB2 receptor active plant metabolites like b-caryophyllene or indirect cannabinoid receptor agonists (FAAH and MAGL inhibitors) in diet may have led to hitherto unrecognized physiological effects. Although it is tempting to believe that these compounds exert benecial effects in humans, clinical evidence is lacking. Future studies will have to determine whether there are additional apparently nontoxic CB2 receptor-selective ligands in plants other than Cannabis and whether they could in fact be exploited therapeutically.

Conict of interest
The authors state no conicts of interest.

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